The present invention relates to the field of molecular and cellular biology. The invention generally relates to methods of synthesizing cDNA. More specifically, the present invention relates to methods of increasing the average cDNA insert size and more particularly, to increasing the percentage of full-length cDNA present within cDNA libraries. Thus, the present invention provides improved cDNA libraries useful in gene discovery.
In examining the structure and physiology of an organism, tissue or cell, it is often desirable to determine its genetic content. The genetic framework of an organism is encoded in the double-stranded sequence of nucleotide bases in the deoxyribonucleic acid (DNA) which is contained in the somatic and germ cells of the organism. The genetic content of a particular segment of DNA, or gene, is only manifested upon production of the protein which the gene encodes. In order to produce a protein, a complementary copy of one strand of the DNA double helix (the “coding” strand) is produced by polymerase enzymes, resulting in a specific sequence of ribonucleic acid (RNA). This particular type of RNA, since it contains the genetic message from the DNA for production of a protein, is called messenger RNA (mRNA).
Within a given cell, tissue or organism, there exist many mRNA species, each encoding a separate and specific protein. This fact provides a powerful tool to investigators interested in studying genetic expression in a tissue or cell. mRNA molecules may be isolated and further manipulated by various molecular biological techniques, thereby allowing the elucidation of the full functional genetic content of a cell, tissue or organism.
A common approach to the study of gene expression is the production of complementary DNA (cDNA) clones. In this technique, the mRNA molecules from an organism are isolated from an extract of the cells or tissues of the organism. This isolation often employs solid chromatography matrices, such as cellulose or agarose, to which oligomers of thymidine (T) have been complexed. Since the 3′ termini on most eukaryotic mRNA molecules contain a string of adenosine (A) bases, and since A binds to T, the mRNA molecules can be rapidly purified from other molecules and substances in the tissue or cell extract. From these purified mRNA molecules, cDNA copies may be made using the enzyme reverse transcriptase (RT) or DNA polymerases having RT activity, which results in the production of single-stranded cDNA molecules. The single-stranded cDNAs may then be converted into a complete double-stranded DNA copy (i.e., a double-stranded cDNA) of the original mRNA (and thus of the original double-stranded DNA sequence, encoding this mRNA, contained in the genome of the organism) by the action of a DNA polymerase. The protein-specific double-stranded cDNAs can then be inserted into a vector, which is then introduced into a host bacterial, yeast, animal or plant cell, a process referred to as transformation or transfection. The host cells are then grown in culture media, resulting in a population of host cells containing (or in many cases, expressing) the gene of interest or portions of the gene of interest.
This entire process, from isolation of mRNA to insertion of the cDNA into a vector (e.g., plasmid, viral vector, cosmid, etc.) to growth of host cell populations containing the isolated gene or gene portions, is termed “cDNA cloning.” If cDNAs are prepared from a number of different mRNAs, the resulting set of cDNAs is called a “cDNA library,” an appropriate term since the set of cDNAs represents a “population” of genes or portions of genes comprising the functional genetic information present in the source cell, tissue or organism. Genotypic analysis of these cDNA libraries can yield much information on the structure and function of the organisms from which they were derived.
The ability to increase the total amount of cDNA produced, and more particularly to produce a cDNA libraries having an increase in the average size of the cDNA molecules and/or to produce cDNA libraries having an increase in the percentage of full-length cDNA molecules would provide a significant advance in cDNA library construction. Specifically, such advances would greatly improve the probability of finding full-length genes of interest.
Ideally, synthesis of a cDNA molecule initiates at or near the 3′ termini of the mRNA molecules. Priming of cDNA synthesis at the 3′ termini at the poly A tail using an oligo(dT) primer ensures that the 3′ message of the mRNAs will be represented in the cDNA molecules produced. Priming which occurs within the mRNA molecules (internal priming) results in synthesis of cDNA molecules which do not contain the full-length message for the genes of interest. That is, internal priming results in truncated cDNA molecules which contain only a portion of the gene or genes of interest. Typically, internal priming causes a loss of the 3′ sequences from the message population. Thus, internal priming lowers the total amount of cDNA produced, decreases the average insert size of cDNA molecules for a cDNA library and/or decreases the percentage of full-length cDNA molecules in a given cDNA library. Sequencing analysis has indicated that many eukaryotic mRNAs have internal poly adenylation stretches which may serve as a priming site when an oligo(dT) primer is used for first strand cDNA synthesis with reverse transcriptase. Moreover, research has shown that some mRNAs can have as many as 16 internal priming sites (Lovett, M., et al., The construction of full-length cDNA libraries by conventional methods and a novel double capture technique, University of Texas Southwestern Medical Center, Dallas, Tex., presented at the 48th Annual Meeting held by The American Society of Human Genetics, Oct. 27-31, 1998, Denver, Colo.). Thus, internal priming of the primer to such internal poly A sequences may adversely affect cDNA synthesis.
The present invention alleviates, prevents, reduces or substantially reduces internal priming thereby providing improvements in cDNA and cDNA library construction. Accordingly, the present invention greatly facilitates gene discovery by providing cDNA libraries containing a greater percentage of full-length genes.
The present invention therefore relates to synthesizing a cDNA molecule or molecules from an mRNA template or population of mRNA templates under conditions sufficient to increase the total amount of cDNA produced, increase the length of the cDNA molecules produced, and/or increase the amount or percentage of full-length cDNA molecules produced. In accordance with the invention, any conditions which inhibit, prevent, reduce or substantially reduce internal priming may be used. Such conditions preferably include but are not limited to optimizing primer concentrations, optimizing reaction temperatures and/or optimizing primer length or specificity. Such result may also be accomplished in accordance with the invention by optimizing the reverse transcription reaction, preferably by inhibiting or preventing reverse transcription until optimum or desired reaction conditions are achieved.
Conventional methods for constructing cDNA libraries use a molar ratio of oligo(dT) primer/mRNA template of 15:1 for first strand cDNA synthesis. The use of such excess amounts of oligo(dT) primer allows internal priming of one or more primers to one or more of the mRNA templates in the reaction. According to a preferred aspect of the present invention, the amount of oligo(dT) primer is reduced for synthesis of first strand cDNA to inhibit, prevent, reduce or substantially reduce internal priming. Preferred molar ratios of primer to template range from about 12:1; 10:1; 9:1; 8:1; 7:1; 6:1; 5:1; 4:1; 3:1; 2:1; 1:1; 1:2; 1:3; 1:4; 1:5; 1:6; 1:7; 1:8; 1:9; 1:10 and 1:12. Preferably, molar ratios of primer (e.g., oligo(dT)) to template (e.g., mRNA) range from about 5:1 to about 1:20, although lower molar ratios of primer to template may be used in accordance with the invention. Specifically, molar ratios of primer to template may be below about 1:10; 1:15; 1:20; 1:25; 1:50; 1:75; and 1:100. Preferably, ranges of molar ratios are below about 5:1; 4:1; 3:1; 2:1; 1:1; 1:2; 1:3; 1:4; and 1:5. Most preferably, ratios of primer to template range from about 10:1 to 1:10; 5:1 to 1:10; 4:1 to 1:10; 3:1 to 1:10; 2.5:1 to 1:10; 2:1 to 1:10; 1.5:1 to 1:10; and 1:1 to 1:10. The optimum ratios of primer to template may vary depending on the primer, mRNA, reverse transcription enzyme and reaction conditions (annealing temperature, buffering salts, etc.). The desired primer to template ratios can be readily determined by one skilled in the art.
In conventional methods of cDNA library construction, annealing or hybridizing primer to template is not carried out at a temperature which prevents, inhibits, reduces or substantially reduces internal priming. Typically, the mixture (e.g., mRNA and oligo(dT) primer) is chilled on ice after denaturation or heating. This process typically causes annealing or hybridization of the primer to internal sites. According to a preferred aspect of the present invention, the temperature during the annealing or hybridization between the primer and the template is maintained so that internal priming is inhibited, prevented, reduced or substantially reduced. In accordance with the invention, such a result is accomplished by carrying out primer annealing or hybridization at higher temperatures. Such conditions may also reduce the formation of mRNA secondary structures during cDNA synthesis. Preferably, temperatures for annealing or hybridizing primers to the templates range from about 10° C. to about 90° C.; more preferably about 10° C. to about 80° C.; still more preferably about 20° C. to about 75° C.; more preferably about 25° C. to about 75° C.; still more preferably about 30° C. to about 65° C.; still more preferably about 37° C. to about 60° C.; still more preferably about 40° C. to about 60° C.; still more preferably about 45° C. to about 60° C.; still more preferably about 45° C. to about 55° C.; and most preferably about 45° C. to about 65° C. The temperature used may vary depending on the type and amount of primer and template and depending on the temperature optimum of the reverse transcription enzyme. The optimum temperature or temperature ranges can be readily determined by one skilled in the art.
Conventional methods for cDNA synthesis typically requires the use of oligo(dT) primers of a particular length (12-18 bases or mer). Such primer length, however, lowers specificity of the primer thereby allowing internal priming. Thus, the invention also relates to increasing specificity of the primers to prevent, inhibit, reduce or substantially reduce internal priming. In a preferred aspect, primer specificity is increased by increasing the length of the primer. Thus, for cDNA synthesis, longer oligo(dT) primers may be used in accordance with the invention. Preferably, primer length ranges from about 20 to about 100 bases, about 20 to about 75 bases, about 20 to about 60 bases, and about 20 to about 50 bases; more preferably about 20 to about 45 bases; more preferably about 20 to about 40 bases; and most preferably about 25 to about 35 bases. In a preferred aspect, the length of the primers are greater than 19 bases; more preferably greater than about 20 bases; more preferably greater than about 25 bases; and still more preferably greater than about 30 bases. Such primer lengths refer to the length of the primers which anneal or hybridize to the template Optimum length and content (nucleotide sequence) of the primers may vary depending on the type of template, the desired reaction conditions, and the reverse transcription enzyme. In accordance with the invention, additional sequences and/or modified nucleotides may be included in the primers of the invention. For example, additional sequences (which do not necessarily anneal or hybridize to the template) may be included in the primers of the invention to assist in cDNA synthesis including sequences comprising one or more restriction endonuclease sites, one or more derivative nucleotides (e.g., hapten containing nucleotides such as biotinylated nucleotides), and the like. The type and length of the primers used in accordance with the invention can be readily determined by one or more skilled in the art.
Conventional cDNA synthesis methods do not control or vary activity of the reverse transcription enzyme to optimize the reverse transcription reaction. In accordance with the invention, the activity of the reverse transcriptase is preferably controlled to start synthesis at a desired time in the reaction. In a preferred aspect, reverse transcriptase activity is inhibited or prevented until optimum or desired reaction conditions are achieved. Such a result is accomplished in accordance with the invention by the use of inhibitors (such as antibodies or antibody fragments) which inhibit reverse transcriptase activity. Such reverse transcriptase inhibitors prevent or inhibit reverse transcriptase activity at low temperatures such that internal priming is prevented, inhibited, reduced or substantially reduced. In accordance with the invention, such inhibitors preferably prevent reverse transcriptase activity below 35° C., below 40° C., below 45° C., below 50° C., below 55° C., below 60° C., below 65° C., below 70° C., below 75° C., below 80° C., below 85° C. and below 90° C. Depending on the thermostability of the enzyme having reverse transcriptase activity, the inhibitor may be designed to inhibit activity of the enzyme at a point at or near the temperature optimum for the enzyme of interest. Preferably, the inhibitor is inactivated at a temperature below or near the temperature optimum of the enzyme used, thereby allowing reverse transcription to take place. Thus, the invention generally relates to the use of reverse transcriptase inhibitors in cDNA synthesis. The type and amount of inhibitor may vary depending on the type and amount of reverse transcription enzyme and depending on the reaction conditions to be used. The type of inhibitor and conditions used with such inhibitor can be readily determined by one of ordinary skill in the art.
In accordance with the invention, any one or a combination of the above improvements to cDNA synthesis may be used. Using any one or a combination of these improvements provides for improved first strand cDNA synthesis (e.g., more total cDNA, larger cDNA and/or more full-length cDNA). In accordance with the invention, the first strand cDNA molecules may be used as templates to make one or more double stranded nucleic acid molecules (e.g., double strand cDNA molecules) by incubating one or more of the first strand cDNA molecules produced by the methods of the invention under conditions sufficient to make one or more nucleic acid molecules complementary to all or a portion of the first strand cDNA molecules. Conditions for making double stranded nucleic acid molecules preferably include incubation with one or more components consisting of one or more DNA polymerases, one or more nucleotides, one or more buffering salts, and one or more primers. In another aspect of the invention, such conditions are modified to provide an increase in the total amount of double stranded cDNA produced, an increase in the length or size of the double stranded cDNA molecule produced, and/or an increase in percentage full-length double stranded cDNA molecule produced. Preferably, such conditions relate to optimization of ribonuclease (RNase) digestion after first strand cDNA synthesis. During first strand cDNA synthesis, if a full-length cDNA molecule complementary to the mRNA template is not made, a single stranded mRNA containing the cap structure will be present at the 5′ end of the mRNA of the mRNA/cDNA hybrid. If a full-length cDNA is produced, a double stranded mRNA/cDNA hybrid is produced with no single stranded mRNA present. Preferably, such digestion conditions are optimized so that the single stranded mRNA of the mRNA/cDNA double stranded molecules formed during first strand cDNA synthesis is subject to RNase digestion. In this manner, cap structure from mRNA/cDNA hybrids which are not full-length are removed while full-length mRNA/cDNA hybrids will retain the cap structure. Thus, cap capture can be used to select for full-length molecules and select against molecules which are not full-length. In a preferred aspect, the conditions are such that the single stranded mRNA of the mRNA/cDNA hybrid is digested or degraded while the mRNA of the double stranded mRNA/cDNA hybrid is not degraded or not substantially degraded. Thus, such RNase digestion is conducted under conditions such that second strand synthesis is not substantially adversely affected. That is, second strand synthesis in accordance with the invention produces larger double stranded cDNA molecules compared to conventional techniques. Conventional RNase I conditions typically range from 25 u/μg to 40 u/μg mRNA at 37° C. and RNase A conditions typically are 1000 ng/μg mRNA at 37° C. Using conventional RNase digestion, the average size of double stranded cDNA molecules produced is about 200 bases. According to the present invention the average size of double stranded cDNA molecules produced is preferably greater than about 300 bases, greater than about 400 bases, greater than about 500 bases, greater than about 600 bases, greater than about 700 bases, greater than about 800 bases, greater than about 900 bases, greater than about 1 kilobase, greater than about 1.5 kilobases, and greater than about 2 kilobases. In one embodiment of the invention, the concentration of the ribonuclease, the type of ribonuclease and reaction conditions are optimized to improve double stranded cDNA synthesis in accordance with the invention. Preferred ribonucleases for use in ribonuclease digestions include ribonuclease A (RNase A) and/or ribonuclease I (RNase I). Generally, lower temperatures (about 4° C. to about 50° C.) and higher salt concentrations (about 5 mM to about 5 M) will assist in inhibiting or controlling RNase digestion in accordance with the invention. Salts used may include sodium chloride, potassium, chloride, magnesium chloride, sodium acetate etc. Additionally, lowering RNase amounts or concentrations may be used to accomplish the desired result. Such concentrations for RNase A may range from about 0.001 ng/μg mRNA to about 500 ng/μg of mRNA and for RNase I may range from about 0.001 u/μg mRNA to about 500 u/μg mRNA. The incubation temperature, RNase concentration and salt concentration may be readily determined by one skilled in the art. In a preferred aspect, concentration of the RNase A include ranges from 0.1 ng/μg mRNA to 10 ng/μg mRNA in TE buffer (10 mM Tris, pH 7.5, 1 mM EDTA) at 37° C. Alternatively, the concentration of the RNase A can include ranges from 0.1 ng/μg mRNA to 500 ng/μg mRNA in 10 mM Tris, pH 7.5 buffer containing 250 mM NaCl at 25° C. for 30 minutes. Preferably, concentration of the RNase I used ranges from 0.1 unit/μg mRNA to 1.0 unit/μg mRNA in 10 mM Tris-HCl (pH 7.5), 5 mM EDTA (pH 8.0), 200 mM sodium acetate at 37° C. Alternatively, the concentration of the RNase I can be used at ranges from 1.0 unit/μg mRNA to 100 units/μg mRNA in the same buffer at 25° C. for 30 minutes.
In another aspect, the invention relates to capture or binding of the cap structure (e.g., m7GpppN) of the mRNA before, during or after first strand cDNA synthesis. Thus, the invention relates to selection of mRNA (before first strand synthesis) or mRNA/cDNA hybrids (after or during first strand synthesis) which have the cap structure in carrying out the methods of the invention. Such selection or capture may be accomplished with any cap binding molecule such as eIF4E, eIF4E peptides, eIF4E peptide fragments (see WO 98/08865) and antibodies or antibody fragments specific for cap structure. In a preferred aspect, selection of the cap structure is accomplished after first strand synthesis. More preferably, such cap capture occurs after ribonuclease digestion in accordance with the methods of the invention. For example, mRNA/cDNA hybrids subjected to ribonuclease digestion are captured and then used for second strand cDNA synthesis according to the invention.
Thus, the present invention is generally directed to methods of synthesizing nucleic acid molecules. The present invention is more specifically directed to methods of making one or more nucleic acid molecules, especially cDNA molecules or cDNA libraries, comprising mixing one or more nucleic acid templates (preferably mRNA, poly A RNA or a population of mRNA molecules) with at least one polypeptide having reverse transcriptase activity, and incubating the mixture under conditions sufficient to make one or more first nucleic acid molecules (e.g., first strand cDNA) complementary to all or a portion of the one or more nucleic acid templates.
In accordance with the invention, such conditions provide for an increased total amount of nucleic acid molecule (cDNA) produced, compared to conventional procedures which do not employ the improved modifications or conditions of the invention. The invention also provides for an increase of length or average size of the nucleic acid molecules (cDNA) produced and/or an increase in the percentage or amount of full-length nucleic acid molecules (cDNA) produced, compared to conventional procedures which do not employ the improved modifications or conditions of the invention. Determining the amount, length and full-length content of the cDNA produced can be determined by conventional techniques well known in the art and as described herein. The percentage or average percentages of full-length cDNA in cDNA libraries produced in accordance with the invention are preferably above about 15%, more preferably above about 20%, more preferably above about 25%, more preferably above about 30%, more preferably above about 40%, more preferably above about 50%, more preferably above about 60%, more preferably above about 70%, more preferably above about 80% and most preferably above about 90% Such full-length percentages are preferably determined by random selection of a portion of the clones of the cDNA library of interest (e.g., 100 to 1000 clones), sequencing the clones and comparing the sequences to known sequence data bases.
In preferred aspects of the invention, the improved results of the invention are preferably accomplished by one or a combination of modifications to the conditions for nucleic acid or cDNA synthesis. Such conditions preferably include modifications for improving first strand cDNA synthesis and/or improving second strand cDNA synthesis.
In a preferred aspect, the invention specifically relates to methods of making one or more double stranded cDNA molecules comprising incubating one or more mRNA molecules preferably a population of mRNA molecules) with one or more primers of the invention at temperatures and primer concentrations to prevent, inhibit, reduce or substantially reduce internal priming prior to or during first strand cDNA synthesis. Such reaction is preferably conducted in the presence of one or more inhibitors of reverse transcriptase activity in accordance with the invention. Ribonuclease digestion is preferably conducted before second strand cDNA synthesis and at ribonuclease concentrations sufficient to increase the length, amount and/or size of double stranded cDNA molecules produced during second strand synthesis. In accordance with the invention, cap capture is preferably accomplished during or after the ribonuclease digestion.
The invention is also directed to nucleic acid molecules and cDNA molecules or populations of cDNA molecules (single or double-stranded) produced according to the above-described methods and to vectors (particularly expression vectors) comprising these nucleic acid molecules and cDNA molecules. The invention also relates to host cells containing such cDNA molecules and/or vectors.
The invention is also directed to kits for use in the methods of the invention. Such kits can be used for making single or double-stranded nucleic acid molecules. The kits of the invention comprise a carrier, such as a box or carton, having therein one or more containers, such as vials, tubes, bottles and the like. Such kits may comprise at least one component selected from the group consisting of primers (preferably primers having higher specificity and most preferably oligo(dT) primers having a length equal to or greater than 20 bases), one or more polypeptides having reverse transcriptase activity (reverse transcriptases and DNA polymerases), one or more inhibitors of reverse transcription (e.g., antibodies and antibody fragments directed against polypeptides having RT activity), one or more cap binding molecules (e.g., antibodies or antibody fragments directed against cap structure), nucleic acid synthesis reaction buffers, one or more nucleotides, one or more vectors, and instructions for carrying out the methods of the invention.
The invention also relates to compositions for use in the invention or made while carrying out the methods of the invention. Such compositions may comprise at least one primer (e.g., oligo(dT) or derivatives thereof) and at least one template in a sample or reaction mixture in amounts or ratios in accordance with the invention. Such composition may further comprise one or more polypeptides having reverse transcriptase activity, one or more reverse transcription inhibitors (e.g., anti-RT antibodies or fragments thereof), one or more nucleotides, one or more cap binding molecules (e.g., anti-cap antibodies for fragments thereof), one or more buffering salts and the like. Such compositions may also be maintained at a temperature to avoid internal priming in accordance with the invention.
The compositions of the invention may also comprise amounts of ribonuclease in accordance with the invention. Such compositions may further comprise at least one component selected from one or more mRNA/cDNA hybrids, one or more nucleotides, one or more polypeptides having reverse transcriptase activity, one or more buffering salts, one or more cap binding molecules (e.g., anti-cap antibodies or fragments thereof) and the like.
The invention also relates to one or more antibodies (monoclonal and polyclonal) and fragments thereof for use in the methods, compositions and kits of the invention. Such antibodies, include anti-cap and/or anti-RT antibodies and antibody fragments
Other preferred embodiments of the present invention will be apparent to one of ordinary skill in the art in view of the following drawings and description of the invention.